Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central domain.

Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.     

Recent independent evolution of msp1 polymorphism in Plasmodium vivax and related simian malaria parasites

The Plasmodium MSP-1 is a promising malaria vaccine candidate. However, the highly polymorphic nature of the MSP-1 gene (msp1) presents a potential obstacle for effective vaccine development. To investigate the evolutionary history of msp1 polymorphism in P. vivax, we construct phylogenetic trees of msp1 from P. vivax and related monkey malaria parasite species. All P. vivax msp1 alleles cluster in the P. vivax lineage and are not distributed among other species. Similarly, all P. cynomolgi msp1 alleles cluster in the P. cynomolgi lineage. This suggests that, in contrast to presumed ancient origin of P. falciparum msp1 polymorphism, the origin of P. vivax msp1 polymorphism is relatively recent. We observed positive selection in the P. vivax lineage but not in P. cynomolgi. Also, positive selection acts on different regions of msp1 in P. vivax and P. falciparum. This study shows that the evolutionary history of msp1 differs greatly among parasite lineages.     

Characterisation of two novel proteins from the asexual stage of Plasmodium falciparum, H101 and H103

The merozoite surface of the pathogenic malaria parasite Plasmodium falciparum is comprised of proteins that are important for the identification and invasion of human red cells. Merozoite surface protein (MSP)3 is a polymorphic protein associated with the surface of merozoites and is also a vaccine candidate. A distinct feature of the MSP3 sequence is three blocks of alanine-rich heptad repeats that are predicted to form an intramolecular coiled-coil. Three orthologues of MSP3 that also contain alanine-rich heptad repeats have been described in P. vivax and we therefore searched the P. falciparum genome database for MSP3 paralogues. We have identified two genes, H101 and H103 related to MSP3, however like another MSP3 paralogue, MSP6, H101 and H103 do not contain heptad repeats. H101 and H103 are expressed during the asexual cycle and immunofluorescence indicates H103 localises to the merozoite surface as a peripheral membrane protein. Transfected parasite lines that express truncated forms of H101 or H103 were viable and grew at the same rate as the parental parasite line. This result may reflect redundancy in function among members of the MSP3/MSP6 gene family as has been described for other families of paralogue genes in P. falciparum.     

Plasmodium vivax merozoite surface proteins-3beta and-3gamma share structural similarities with P. vivax merozoite surface protein-3alpha and define a new gene family

The genes encoding two merozoite surface proteins of Plasmodium vivax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. vivax lambdagt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. vivax lambdaDASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75677 Da, and 3' and 5' RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. vivax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. vivax genes have putative signal peptides, large central domains that contain >20% alanine residues bound by charged regions, are predicted to form alpha-helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low (<40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3alpha, Pvmsp-3beta, and Pvmsp-3gamma. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparum.     

Natural variation within the principal adhesion domain of the Plasmodium vivax duffy binding protein.

The blood-stage development of malaria parasites is initiated by the invasion of merozoites into susceptible erythrocytes. Specific receptor-ligand interactions must occur for the merozoites to first attach to and then invade erythrocytes. Because the invasion process is essential for the parasite's survival and the merozoite adhesion molecules are exposed on the merozoite surface during invasion, these adhesion molecules are candidates for antibody-dependent malaria vaccines. The Duffy binding protein of Plasmodium vivax belongs to a family of erythrocyte-binding proteins that contain functionally conserved cysteine-rich regions. The amino cysteine-rich regions of these homologous erythrocyte-binding proteins were recently identified for P. vivax, Plasmodium knowlesi, and Plasmodium falciparum as the principal erythrocyte-binding domains (C. Chitnis and L. H. Miller, J. Exp. Med. 180:497-506, 1994, and B. K. L. Sim, C. E. Chitnis, K. Wasniowska, T. J. Hadley, and L. H. Miller, Science 264:1941-1944, 1994). We report that amino acids in this critical ligand domain of the P. vivax Duffy binding protein are hypervariable, but this variability is limited. Hypervariability of the erythrocyte-binding domain suggests that this domain is the target of an effective immune response, but conservation of amino acid substitutions indicates that functional constraints limit this variation. In addition, the amino cysteine-rich region and part of the hydrophilic region immediately following it were the site of repeated homologous recombinations as represented by tandem repeat sequence polymorphisms. Similar polymorphisms have been identified in the same region of the homologous genes of P. falciparum and P. knowlesi, suggesting that there is a common mechanism of recombination or gene conversion that occurs in these Plasmodium genes.     

Structural studies on Plasmodium vivax merozoite surface protein-1

Plasmodium vivax infection is the second most common cause of malaria throughout the world. Like other Plasmodium species, P. vivax has a large protein complex, MSP-1, located on the merozoite surface. The C-terminal MSP-1 sub-unit, MSP-1(42), is cleaved during red blood cell invasion, causing the majority of the complex to be shed and leaving only a small 15kDa sub-unit, MSP-1(19), on the merozite surface. MSP-1(19) is considered a strong vaccine candidate. We have determined the solution structure of MSP-1(19) from P. vivax using nuclear magnetic resonance (NMR) and show that, like in other Plasmodium species, it consists of two EGF-like domains that are oriented head-to-tail. The protein has a flat, disk-like shape with a highly charged surface. When MSP-1(19) is part of the larger MSP-1(42) precursor it exists as an independent domain with no stable contacts to the rest of the sub-unit. Gel filtration and analytical ultracentrifugation experiments indicate that P. vivax MSP-1(42) exists as a dimer in solution. MSP-1(19) itself is a monomer, however, 35 amino-acids immediately upstream of its N-terminus are sufficient to cause dimerization. Our data suggest that if MSP-1(42) exists as a dimer in vivo, secondary processing would cause the dissociation of two tightly linked MSP-1(19) proteins on the merozoite surface just prior to invasion.     

Antibodies to lactate dehydrogenase of Plasmodium knowlesi are specific to Plasmodium species

Polyclonal immune monkey serum raised against schizonts of Plasmodium knowlesi (H-strain) showed the presence of antibodies to lactate dehydrogenase (LDH) of P. knowlesi by immunodot enzyme staining method. The anti-LDH antibodies are most probably directed towards an epitope distinct from the catalytic site as shown by the specific enzyme staining of LDH after binding with antibody on nitrocellulose paper. These antibodies showed reactivity with LDH from different strains (H, P and W1 strains of P. knowlesi) and species (P. cynomolgi B, P. berghei, P. yoelii, P. falciparum and P. vivax) of malarial parasites but did not cross-react with three isoenzymic forms of mammalian LDH (A4, B4 and C4) as well as with LDH from some protozoan and helminth parasites. These findings suggest that the anti-LDH antibodies have defined specificity to Plasmodium spp.     

Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion

Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi alpha protein, which binds human and rhesus Duffy antigens, and P. knowlesi beta and gamma proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here, we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of erythrocyte binding proteins. Region II of P. knowelsi alpha protein (Pk(alpha)RII), which binds the Duffy antigen, was expressed as a secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant Pk(alpha)RII were tested for inhibition of erythrocyte binding and invasion. Antibodies raised against Pk(alpha)RII inhibit P. knowlesi invasion of both human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175.     

The Plasmodium vivax homologues of merozoite surface proteins 4 and 5 from Plasmodium falciparum are expressed at different locations in the merozoite

Merozoite surface proteins of Plasmodium falciparum are one major group of antigens currently being investigated and tested as malaria vaccine candidates. Two recently described P. falciparum merozoite surface antigens, MSP4 and MSP5, are GPI-anchored proteins that each contain a single EGF-like domain and appear to have arisen by an ancient gene duplication event. The genes are found in tandem on chromosome 2 of P. falciparum and the syntenic region of the genome was identified in the rodent malarias P. chabaudi, P. yoelii and P. berghei. In these species, there is only a single gene, designated MSP4/5 encoding a single EGF-like domain similar to the EGF-like domain in both PfMSP4 and PfMSP5. Immunization of mice with PyMSP4/5 provides mice with high levels of protection against lethal challenge with blood stage P. yoelii. In this study, we show that in P. vivax, which is quite phylogenetically distant from P. falciparum, both MSP4 and MSP5 homologues can be found with their relative arrangements with respect to the surrounding genes mostly preserved. However, the gene for MSP2, found between MSP5 and adenylosuccinate lyase (ASL) in P. falciparum, is absent from P. vivax. The PvMSP4 and PvMSP5 genes have a two-exon structure and encode proteins with potential signal and GPI anchor sequences and a single EGF-like domain near the carboxyl-terminus. Rabbit antisera raised against purified recombinant proteins show that each of the antisera react with distinct proteins of 62 kDa for PvMSP4 and 86 kDa for PvMSP5 in parasite lysates. Indirect immunofluorescence assays (IFA) localized PvMSP4 over the entire surface of P. vivax merozoites, as expected, whereas, the MSP5 homologue was found to be associated with an apical organellar location consistent with micronemes or over the polar prominence.     

Cloning of the Plasmodium vivax Duffy receptor.

Plasmodium vivax and Plasmodium knowlesi merozoites invade only Duffy blood group-positive human erythrocytes. Soluble P. vivax and P. knowlesi merozoite proteins of 135 kDa bind specifically to Duffy blood group determinants. The gene encoding a member of the Duffy receptor gene family of P. knowlesi has been cloned. We report here the molecular cloning of the presumptive Duffy receptor gene of P. vivax, using the P. knowlesi gene as a probe. There is a single gene in P. vivax which codes for a protein of 1115 amino acids. The deduced amino acid sequence predicts a putative signal sequence at the amino-terminus and a transmembrane region followed by 45 amino acids at the carboxy-terminus. The three introns found at the 3' end of the P. knowlesi gene were conserved in P. vivax, including high homology for the sequences of the introns. Comparison of the portion of the proteins amino to the transmembrane region between P. vivax and the partial sequence of P. knowlesi indicated at least three domains. Two homologous regions were separated by a non-homologous region. The cysteines in the homologous regions were conserved in number and position, indicating that the folding is similar and suggesting that these regions may be the Duffy blood group binding domains. In both P. vivax and P. knowlesi, the non-homologous region is hydrophilic and proline-rich, although the position of the prolines is not conserved. As prolines tend to stiffen a protein, this region may act as a 'hinge region' similar to those in the immunoglobulin gene family.     

Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys

The 42-kDa fragment of the merozoite surface protein 1 (MSP-1(42)) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-1(42) (PvMSP-1(42)) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-1(42) protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-1(42) protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-1(42) (PcMSP-1(42)) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-1(42)-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-1(42) group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm(-3). Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-1(42) and PvMSP-1(42) proteins also appeared to have a similar partially protective effect.     

Mass spectrometric identification of an HLA-A*0201 epitope from Plasmodium falciparum MSP-1

Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum-derived antigens were shown to play an important role for the protection against malaria. Although several CTL epitopes have been identified from P. falciparum sporozoite-derived antigens, none has been described for the merozoite form. Since the merozoite surface protein (MSP)-1 is a known target of the immune response, we focused on this protein to identify HLA-A*0201-associated epitopes. Using our mass spectrometry-based method [the 'predict-calibrate-detect' (PCD) approach], we were able to identify an MSP-1-derived epitope in the peptide mixture naturally associated with HLA-A*0201 molecules purified from an MSP-1-expressing cell line. CTLs against this epitope were generated from HLA-A*0201 monochain transgenic mice (HHD). They specifically killed MSP-1-expressing HLA-A2-positive target cells. Thus, we describe here the first MHC class I epitope from the merozoite form of P. falciparum. This epitope can be used as a tool for the immunomonitoring of natural or vaccine-induced CTL immune responses against malaria and could eventually be proposed as a component of an anti-malaria peptide-based vaccine.     

A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain.

Merozoite surface proteins of Plasmodium falciparum play a critical role in the invasion of human erythrocytes by the malaria parasite. Here we describe the identification of a novel protein with a molecular mass of 40 kDa that is found on the merozoite surface of P. falciparum. We call this protein merozoite surface protein 4 (MSP-4). Evidence for the surface location of MSP-4 includes (i) a staining pattern that is consistent with merozoite surface location in indirect immunofluorescent studies of cultured parasites, (ii) localization of MSP-4 in the detergent phase in Triton X-114 partitioning studies, and (iii) nucleotide sequencing studies which predict the presence of an N-terminal signal sequence and a hydrophobic C-terminal sequence in the protein. Immunoprecipitation studies of biosynthetically labelled parasites with [3H] myristic acid indicated that MSP-4 is anchored on the merozoite surface by a glycosylphosphatidylinositol moiety. Of considerable interest is the presence of a single epidermal growth factor-like domain at the C terminus of the MSP-4 protein, making it the second protein with such a structure to be found on the merozoite surface.     

Amino terminus of Plasmodium falciparum acidic basic repeat antigen interacts with the erythrocyte membrane through band 3 protein

The acidic basic repeat antigen (ABRA) of Plasmodium falciparum is localised in the parasitophorous vacuole, and associates with the merozoite surface at the time of schizont rupture. By virtue of its protease-like activity, it is implicated in the process of merozoite invasion and schizont rupture, and therefore, possibly interacts with erythrocyte membrane proteins to execute its function during these events. In this study, using Escherichia coli expressed recombinant fragments of ABRA, we have demonstrated that ABRA interacts with red blood cells through its N-terminus. Out of the four human erythrocyte proteins tested, namely, band 3, glycophorin A and B and spectrin, ABRA showed dose-dependent and saturable binding with the band 3 protein. This binding was lost on chymotrypsin treatment of erythrocytes or their membrane extract. Studies with the deletion constructs of the N-terminus revealed that the binding domain lies in the cysteine-rich N-proximal region of ABRA. In addition to the recombinant fragments, native ABRA derived from the P. falciparum-infected erythrocytes also showed binding to band 3 protein. Sequencing of the cysteine-rich 528 bp region, amplified from fifteen field isolates of P. falciparum, showed that not only the five cysteines of mature ABRA but also the whole sequence is fully conserved, even at the nucleotide level. This sequence conservation of the N-terminus and its role in RBC binding suggests that this region may be crucial for any putative function of ABRA, therefore emphasising its importance as a vaccine/drug target.     

Use of Stable Sensitized Cells in an Improved Indirect Microhemagglutination Test for Malaria

Aldehyde-fixed cells sensitized with Plasmodium knowlesi and P. falciparum antigens were tested with sera from P. vivax and P. falciparum infections. Titers were improved by use of a mixture of cells sensitized with the two antigens.     

Expression and serologic activity of a soluble recombinant Plasmodium vivax Duffy binding protein.

Plasmodium vivax Duffy binding protein (DBP) is a conserved functionally important protein. P. vivax DBP is an asexual blood-stage malaria vaccine candidate because adhesion of P. vivax DBP to its erythrocyte receptor is essential for the parasite to continue development in human blood. We developed a soluble recombinant protein of P. vivax DBP (rDBP) and examined serologic activity to it in residents of a region of high endemicity. This soluble rDBP product contained the cysteine-rich ligand domain and most of the contiguous proline-rich hydrophilic region. rDBP was expressed as a glutathione S-transferase (GST) fusion protein and was isolated from GST by thrombin treatment of the purified fusion protein bound on glutathione agarose beads. P. vivax rDBP was immunogenic in rabbits and induced antibodies that reacted with P. vivax and Plasmodium knowlesi merozoites. Human sera from adult residents of a region of Papua New Guinea where malaria is highly endemic or P. vivax-infected North American residents reacted with rDBP in an immunoblot and an enzyme-linked immunosorbent assay. The reactivity to reduced, denatured P. vivax rDBP and the cross-reactivity with P. knowlesi indicated the presence of immunogenic conserved linear B-cell epitopes. A more extensive serologic survey of Papua New Guinea residents showed that antibody response to P. vivax DBP is common and increases with age, suggesting a possible boosting of the antibody response in some by repeated exposure to P. vivax. A positive humoral response to P. vivax DBP correlated with a significantly higher response to P. vivax MSP-1(19). The natural immunogenicity of this DBP should strengthen its usefulness as a vaccine.     

Lack of sequence diversity in the gene encoding merozoite surface protein 5 of Plasmodium falciparum.

The gene encoding merozoite surface protein 5 (MSP5) of Plasmodium falciparum is situated between the genes encoding MSP2 and MSP4 on chromosome 2. Both MSP4 and MSP5 encode proteins that contain hydrophobic signal and glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. The similar gene organization, location and similar structural features of the two genes suggest that they have arisen from a gene duplication event. In this study we provide further evidence for the merozoite surface location of MSP5 by demonstrating that MSP5 is present in isolated merozoites, partitions in the detergent-enriched phase following Triton X-114 fractionation and shows a staining pattern consistent with merozoite surface location by indirect immunofluorescence confocal microscopy. Analysis of antigenic diversity of MSP5 shows a lack of sequence variation between various isolates of P. falciparum from different geographical locations, a feature unusual for surface proteins of merozoites and one that may simplify vaccine formulation.     

Characterization of the merozoite surface protein 4/5 gene of Plasmodium berghei and Plasmodium yoelii

The genes encoding merozoite surface protein 4/5 (MSP4/5) from Plasmodium berghei and Plasmodium yoelii have been cloned and completely sequenced. Comparisons of the predicted protein sequences with those of Plasmodium chabaudi MSP4/5 and Plasmodium falciparum MSP4 and MSP5 show general structural similarities. All predicted proteins contain hydrophobic signal sequences, potential GPI attachment sequences and a single epidermal growth factor (EGF)-like domain at the C-terminus. The amino acid sequence of the EGF-like motif is highly conserved in rodent malaria species and also shows a considerable degree of similarity with the EGF-like domains found in the P. falciparum proteins. Both the P. yoelii and P. berghei genes show evidence of both spliced and unspliced mRNA at steady state. This phenomenon is similar to that seen for the P. chabaudi MSP4/5 gene, and is believed to be involved in regulation of protein expression. We describe here the construction of clones expressing full length recombinant protein. Antibodies directed against recombinant MSP4/5 proteins recognize a single polypeptide on parasite material and show crossreactivity between MSP4/5 from different murine malaria species, but do not crossreact with either MSP4 or MSP5 from P. falciparum. The various antisera show reactivity against reduction sensitive epitopes as well as reduction insensitive epitopes.     

Interaction between two domains of the P. yoelii MSP-1 protein detected using the yeast two-hybrid system

Several model systems of plasmodia have demonstrated the potential of the merozoite surface protein, MSP-1, to induce protective immunity. However, little is known about the function of this protein or its interaction with other surface molecules that may also serve as immunological targets. To identify potentially significant inter- and intra-molecular interactions involving MSP-1, we have utilized the yeast two-hybrid system. A cDNA activation domain library was constructed from the erythrocytic stages of the murine malarial parasite Plasmodium yoelii yoelii 17XL. A 795 bp region of Py17XL MSP-1 (bait), homologous to the Plasmodium falciparum MSP1(33) fragment, was inserted into a Gal4p DNA binding domain vector and used to screen the activation domain library (target). Several randomly selected clones that demonstrated bait-target interaction were found to express overlapping regions of Py17XL MSP-1. Deletion constructs further localized the peptide fragments retaining interaction indicating that a region within the MSP-1(38) fragment interacts with the MSP-1 bait domain. Subsequent studies confirmed this interaction, as both peptides were co-precipitated from cell lysate by a peptide tag-specific antibody. It was observed that the interaction of these two fragments significantly increased the half-life of the MSP-1(38) within yeast cells. The specific interaction described here demonstrates the potential of this approach to elucidate additional inter- or intra-molecular interactions of Py17XL MSP1 and other malarial proteins.     

Identification of proteins from Plasmodium falciparum that are homologous to reticulocyte binding proteins in Plasmodium vivax

Plasmodium falciparum infections can be fatal, while P. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereas P. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5' end but are highly divergent at the 3' end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.